A plant/fungal-type phosphoenolpyruvate carboxykinase located in the parasite mitochondrion ensures glucose-independent survival of Toxoplasma gondii

Abstract

is considered to be one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host cell types of essentially any warm-blooded organism. Our earlier work demonstrated that the acute (tachyzoite) stage of depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not yet been resolved. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in theiochondrion (PEPCK), whereas the other protein is otxpressed in achyzoites (PEPCK). Parasites with an intact glycolysis can tolerate genetic deletions ofPEPCK as well as of PEPCK, indicating their nonessential roles for tachyzoite survival.PEPCK can also be ablated in a glycolysis-deficient mutant, while PEPCK is refractory to deletion. Consistent with this, the lytic cycle of a conditional mutant of PEPCK in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCK in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function ofPEPCK in cohesive operation of glycolysis and the tricarboxylic acid cycle in a normal glucose-replete milieu. Conversely, we found that the otherwise integrative enzyme pyruvate carboxylase (PyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.

The intracellular parasite Toxoplasma gondii harbors three druggable FNT-type formate and l-lactate transporters in the plasma membrane

“…S4). An apparent redundancy in L-lactate transport is also in accordance with the exceptional metabolic plasticity observed in the central carbon metabolism of tachyzoites (16,18,19). FNTs discriminate between different monocarboxylate substrates by size selection in the ⌽/K filter region (7).…”

“…Earlier work has demonstrated the expression of high-affinity sugar permeases in both parasites (12,13), which allow import of glucose (besides other hexoses) and its subsequent catabolism through glycolysis (14 -16). Surprisingly, glucose import is essential for asexual growth of the acute (termed merozoite) stage in Plasmodium (17,18) but dispensable for the acute (termed tachyzoite) stage of T. gondii (16,18,19). Previous works by several groups have also characterized lactate dehydrogenases in the two parasites (20,21), which are meant to enable recycling of NADH ϩ to NAD ϩ and thus ensure a continued glycolysis.…”

Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability

“…Several bacterial species are known to degrade taurine via a pathway that results in the nitrogen being incorporated into alanine and other amino acids [24,25]. However, no 15 N was detected in any intracellular metabolite except taurine, and taurine levels did not decrease as previously observed. This…”

“…This glutamine to glucose pathway relies on a gluconeogenic enzyme fructose bisphosphatase 2, which was found to be constitutively expressed and essential to growth [7]. Similarly, T. gondii catabolism of glutamine to fuel gluconeogenesis is reliant on a mitochondrial phosphoenolpyruvate carboxykinase enzyme, which is thought to play a key regulatory role in T. gondii carbon metabolism [15]. The metabolic flexibility of T. gondii is remarkable, recent work has even shown that growth will occur in environments completely lacking glucose and glutamine [16].…”

An unusual and physiologically vital protein with guanylate cyclase and P-type ATPase like domain in a pathogenic protist

“…Genomic-tagging of TgATPase P -GC and TgPKG--Sequences of TgATPase P -GC (TGGT1_254370) and TgPKG (TGGT1_311360) genes were obtained from the parasite genome database (ToxoDB) (Gajria, Bahl, Brestelli, Dommer, Fischer, Gao, Heiges, Iodice, Kissinger, Mackey, Pinney, Roos, Stoeckert, Wang and Brunk 2008). The expression and subcellular localization of TgATPase P -GC and TgPKG were determined by 3'-insertional tagging (3'IT) of corresponding genes with an epitope, essentially as reported before (Nitzsche et al 2017). To achieve this, the 3' end of the gene (1-1.5 kb 3'-crossover sequence or COS) was amplified from gDNA of the RHΔku80-hxgprtstrain using Q5 TM High-Fidelity DNA Polymerase (Bio-Rad Laboratories, Germany) (see Table S1 for primers).…”